Most LO inhibitors work through an antioxidant action in the enzyme active site. A recent report (8) indicated that cannabinoids such as THC and cannabidiol are ...
CANNABIDIOL IS A SUBTYPE-SPECIFIC INHIBITOR OF INFLAMMATORY LIPOXYGENASE ENZYMES. Aidan J Hampson AIMS This study examines cannabidiol as an inhibitor of 5-, and 15-LO’s, both at the enzyme level and in whole human blood cells. The nature of cannabidiol inhibition is also be examined.
Cannabidiol inhibits 15-LO but not 12-LO
12-HETE formation (% ctrl)
% abs change (234 nM)
Introduction
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0 -7
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CBD inhibition decreases as substrate concentration increases. Furthermore, HPLC analysis indicated CBD is not a substrate for 15-LO
Effect of CBD on 12-HETE formation (12-LO product) in human leukocytes
100
PlA2 inhibitor No PlA2 inhibitor -5.0
Conclusions
-5.0
-4.5
Platelet LO is not related to leukocyte-LO. Leukocyte 12-LO is closely related to 15-LO. However, CBD inhibits 15-LO but not 12-LO’s. Analysis was performed by HPLC
12-HETE protects neurons from AMPAr toxicity
12-HETE protects cortical neurons from NMDAr toxicity References
% of ctrl LDH production
75
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1. 2.
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Steinhilber D: Curr Med Chem 1999 6:71 Burstein SH et al: J Pharmacol Exp Ther 1989 251: 531
4.
Evans TA et al: Biochem Pharmacol 1987 36: 2035
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White HL; Tansik RL: Prostaglandins Med 1980 4:409
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Audette CA; Burstein SH:Life Sci 1990 47: 753
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Samuelsson B: Adv Exp Med Biol 1997 433:1
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Diaz S et al: J Pharmacol Exp Ther 1994 268: 1289
8.
Hampson AJ et al: Proc Natl Acad Sci U S A 1998 95: 8268
9.
Yamamoto S et al: Prog Lipid Res 1997 36:23
10. Murphy E et al: Circ Res 1995 76:457 11. Piomelli D; GreenGard P Trends Pharmacol Sci 1990 11:367
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25 100glu
4. 12-LO is known to be activated following ischaemic damage, (10) suggesting that part of the protective effect of cannabidiol may be due to its enhancing effect on 12-HETE formation.
5-LO is a major inflammatory enzyme. RBL-2H3 cells were incubated with 10M arachidonic acid and CBD. Samples were analysed by rp-HPLC and peak areas calculated (=236 nM).
PlA2 inhibitor No PlA2 inhibitor
100
% ctrl death
3. 12-HETE, the product of 12-LO is neuroprotective in vitro in both AMPAr and NMDAr dependent glutamate toxicity assays.
-4.0
100
100
2. Cannabinoids stimulate on phospholipase A2 activity, which, when combined with blockade of inflammatory LO’s, results in cannabidiol potentiating 12-LO product formation.
-4.5
CBD concn (M)
Human leukocytes incubated with arachidonic acid CBD and Phospholipase A2 inhibitor (80 M manoalide). CBD increases 12-HETE formation by activating PlA2 while having no effect on 12-LO.
1. Cannabidiol competitively inhibits 5- and 15-type enzymes, but has no effect on 12-LO activity.
-5.0
200
0 -5.5
-4.5
-5.5
Effect of CBD on human platelet 12-LO
12-HETE formation (% ctrl)
12-HETE formation (% ctrl)
200
0 -5.5
-6.0
CBD concn (M)
CBDconcn [M]
CBD concn (M)
The confusion created by apparently conflicting earlier reports, reflects the specificity of cannabidiol and other cannabinoids as lipoxygenase inhibitors.
0 -6.5
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Substrate concn [M]
Rate of LO product was examined by uv Spectrocopy. Absorption at 234 – 238 nM represents formation of a conjugated diene, an indicator of LO activity. EC50= 550nM, Substrate = 10M linoleic acid
6
50
EC50= 1.92 M
Porcine 12-LO Rabbit 15-LO
Unfortunately, the existing knowledge base concerning the anti-inflammatory effects of cannabinoids is somewhat imperfect. Some studies have indicated that cannabinoids inhibit lipoxygenases (LO’s; 3,4), although others have reported no inhibition (5) and still other reports describe cannabinoids as activators of LO dependent pathways (6,7).
Inflammation and oedema are substantial components of many neuropathologies including Stroke, head trauma and possibly AIDS dementia. It is possible therefore, that some of the neuroprotective properties of cannabidiol can be accounted for through its effects on eicosanoid production.
50
50
Over the last twenty years, various reports have suggested that cannabinoids may affect immune cell function and inflammation by altering eicosanoid production. In particular, reports have suggested that cannabinoids inhibit lipoxygenases (LO’s), which produce potent inflammatory mediators (1) associated with many pathologies including; arthritis, asthma, atherosclerosis (2).
Most LO inhibitors work through an antioxidant action in the enzyme active site. A recent report (8) indicated that cannabinoids such as THC and cannabidiol are powerful antioxidants, which may explain their alleged effects as LO inhibitors. This same report indicated that, due to these antioxidant properties, non-psychoactive cannabinoids such as cannabidiol appear to be good (in vitro) neuroprotectants.
Effect of CBD on 5-LO activity (RBL-2H3) 100
% inhibition by 1uM CBD
100
The consequences of cannabidiol’s selective LO inhibition is illustrated. It is demonstrated that non-inflammatory 12-LO products, which are unaffected by cannabidiol, can markedly protect cultured neurons from glutamate toxicity
Furthermore, while cannabinoids may inhibit LO’s, they also cause release of LO substrates through phospholipase A2 activation.
Cannabidiol inhibition is competitive 100
0.03
0.1
0.3
1
3
12-HETE concn [uM]
Cortical neurons were exposed to glutamate (plus MK-801 and cyclothiazide) for 20 hours 12-HETE
Ctrl
during ischemia
post ischemia
During & Post
(0.5ug / ml) 12-HETE was applied both during glutamate insult and afterwards (“reperfusion” phase)
